1. Field of the Invention
The present invention relates generally to liquid chromatography and mass spectrometry. More particularly, the present invention relates to detection and quantification of ions from data collected by an LC/MS system and subsequent or real-time analysis of such data.
2. Background of the Invention
Mass spectrometers (MS) are well-known scientific instruments used widely for identifying and quantifying molecular species in a sample. During analysis, molecules from the sample are ionized to form ions that are introduced into the mass spectrometer for analysis. The mass spectrometer measures the mass-to-charge ratio (m/z) and intensity of the introduced ions.
Mass spectrometers are limited in terms of the number of ions they can reliably detect and quantify within a single spectrum. As a result, samples containing many molecular species may produce spectra that are too complex for interpretation or analysis using conventional mass spectrometers.
In addition, the concentration of molecular species can vary over a wide range. In biological samples, for example, there are typically a greater number of molecular species at lower concentrations than at higher concentrations. Thus, a significant fraction of ions appear at low concentration. The low concentration is typically near the detection limit of the mass spectrometer. Moreover, at low concentration, ion detection also suffers from the presence of background noise and/or the presence of interfering background molecules. Consequently, detecting such low abundance species can be improved by removing as much of the background noise as possible and reducing the number of interfering species that are present in the spectrum at any one time.
A common technique used to reduce the complexity of such spectra is to perform a chromatographic separation prior to injecting the sample into the mass spectrometer. For example, peptides or proteins often produce clusters of ions that elute at a common time and that overlap in spectra. Separating the clusters from the different molecules in time simplifies interpretation of the spectra produced by such clusters.
Instruments commonly used to carry out such chromatographic separation include gas chromatographs (GCs) or liquid chromatographs (LCs). When coupled to a mass spectrometer, the resulting systems are referred to as GC/MS or LC/MS systems respectively. GC/MS or LC/MS systems are typically on-line systems in which the output of the GC or LC is coupled directly to the MS.
A combined LC/MS system provides an analyst with a powerful means to identify and to quantify molecular species in a wide variety of samples. Typical samples can contain a mixture of a few or thousands of molecular species. The molecules themselves can exhibit a wide range of properties and characteristics. For example, each molecular species can produce more than one ion. This can be seen in peptides where the mass of the peptide depends on the isotopic forms of its nuclei; and in the families of charge states into which an electrospray interface can ionize peptides and proteins.
In an LC/MS system, a sample is injected into the liquid chromatograph at a particular time. The liquid chromatograph causes the sample to elute over time resulting in an eluent that exits the liquid chromatograph. The eluent exiting the liquid chromatograph is continuously introduced into the ionization source of the mass spectrometer. As the separation progresses, the composition of the mass spectrum generated by the MS evolves and reflects the changing composition of the eluent.
At regularly spaced time intervals, a computer-based system samples and records the spectrum on a storage device, such as a hard-disk drive. In conventional systems, these acquired spectra are analyzed after completion of the LC separation.
After acquisition, conventional LC/MS systems generate one-dimensional spectra and chromatograms. The response (or intensity) of an ion is the height or area of the peak as seen in either the spectrum or the chromatogram. To analyze spectra or chromatograms generated by conventional LC/MS systems, peaks in such spectra or chromatograms that correspond to ions must be located or detected. The detected peaks are analyzed to determine properties of the ions giving rise to the peaks. These properties include retention time, mass-to-charge ratio and intensity. Mass or mass-to-charge ratio (m/z) estimates for an ion are derived through examination of a spectrum that contains the ion. Retention time estimates for an ion are derived by examination of a chromatogram that contains the ion. The time location of a peak apex in a single mass-channel chromatogram provides an ion's retention time. The m/z location of a peak apex in a single spectral scan provides the ion's m/z value.
A conventional technique for detecting ions using an LC/MS system is to form a total ion chromatogram (TIC). Typically, this technique is applied if there are relatively few ions requiring detection. A TIC is generated by summing, within each spectral scan, all responses collected over all m/z values and plotting the sums against scan time. Ideally, each peak in a TIC corresponds to a single ion.
One problem with this method of detecting peaks in a TIC is possible co-elution of peaks from multiple molecules. As a result of such co-elution, each isolated peak seen in the TIC may not correspond to a unique ion. A conventional method for isolating such co-eluted peaks is to select the apex of one peak from the TIC and collect spectra for the time corresponding to the selected peak's apex. The resulting spectral plot is a series of mass peaks, each presumably corresponding to a single ion eluting at a common retention time.
For complex mixtures, co-elution also typically limits summing of spectral responses to sums only over a subset of collected channels, e.g., by summing over a restricted range of m/z channels. The summed chromatogram provides information about ions detected within the restricted m/z range. In addition, spectra can be obtained for each chromatographic peak apex. To identify all ions in this manner, multiple summed chromatograms are generally required.
Another difficulty encountered with peak detection is detector noise. A common technique for mitigating detector noise effects is to signal-average spectra or chromatograms. For example, the spectra corresponding to a particular chromatographic peak can be co-added to reduce noise effects. Mass-to-charge ratio values as well as peak areas and heights can be obtained from analyzing the peaks in the averaged spectrum. Similarly, co-adding chromatograms centered on the apex of a spectral peak can mitigate noise effects in chromatograms and provide more accurate estimates of retention time as well as chromatographic peak areas and heights.
Aside from these problems, additional difficulties are encountered when conventional peak detection algorithms are used to detect chromatographic or spectral peaks. If performed manually, such conventional methods are not only subjective, but are also quite tedious. Even when performed automatically, such methods can be subjective due to, for example, the subjective choices for thresholds to use to identify peaks. Further, these conventional methods tend to be inaccurate because they analyze data using only a single extracted spectrum or chromatogram, and do not provide ion parameter estimates having the highest statistical precision or lowest statistical variance. Finally, conventional peak-detection techniques do not necessarily provide uniform, reproducible results for ions at low concentration, or for complex chromatograms, where co-elution and ion interference tend to be common problems.